This invention relates to cardiac valve replacement.
Cardiac valve diseases are prevalent clinical problems, usually requiring prosthetic replacement. Valves can become diseased or damaged from a variety of causes. Congenital defects may result in abnormally formed valves. Infections such as rheumatic fever and bacterial endocarditis can lead to valve damage.
The first prosthetic valvular device was implanted in 1952, and a variety of mechanical, bioprosthetic, and homograft valves are presently in use. Thromboembolic events and sudden structural failure are problems associated with traditional mechanical valves. Bioprosthetic xenograft replacement valves have been developed to reduce the risk of such problems. Xenograft valves are typically porcine or bovine. However, such valves are limited in their durability, as calcification and fibrotic sheath formation often lead to stenosis and regurgitation, with a 40% reoperation rate 8-10 years after implantation. Homograft valve transplants are limited by immune and inflammatory recipient responses, limited donor cell viability, and complex matrix issues resulting in degradation of mechanical performance properties.
The invention provides an improved replacement cardiac valve. The bioprosthetic heart valve contains an acellular matrix as a structural scaffold and isolated myofibroblasts. The acellular matrix is preferably an acellular homograft, an acellular xenograft, or a synthetic matrix. The matrix is contacted with isolated myofibroblasts, which are allowed to cellularize the matrix. The myofibroblasts are resistant to dedifferentiation during culture prior to implantation and after implantation into a recipient individual. At least 60% of the total collagen produced by the myofibroblasts is type I collagen. Preferably, the myofibroblasts produce at least 2-fold more type I collagen compared to type III collagen. Reduced type III collagen production is critical to minimizing scar tissue formation in the replacement valve recipient. Accordingly, less than 25%, more preferably less than 20%, and most preferably less than 15% of total collagen production by valve myofibroblasts is type III collagen.
In addition to increased type I collagen production, the myofibroblasts secrete extracellular matrix components, including but not limited to, fibronectin, elastin, and glycosaminoglycans, such as chondroitin sulfate or hyaluronic acid. The myofibroblast cells are cultured in the presence of factors which inhibit dedifferentiation. The cells are cultured in the presence or absence of an acellular matrix or scaffold. For example, the cells are maintained in an endothelial cell-conditioned media, or grown in the presence of endothelial cells. The two cell types may be in direct contact with one another, e.g., in a coculture, or separated by a membrane which allows diffusion of soluble factors but prevents cell-to-cell contact.
The term xe2x80x9cisolatedxe2x80x9d used in reference to a particular cell type, e.g., a myofibroblast or endothelial cell, means that the cell is substantially free of other cell types or compositions with which it naturally occurs. For example, isolated myofibroblasts are obtained from solid heart leaflet tissue but are separated from other cell types which are present in heart leaflet interstitial tissue. Cells are xe2x80x9cisolatedxe2x80x9d when the particular cell type is at least 60% of a cell population. Preferably, the cells represent at least 75%, more preferably at least 90%, and most preferably at least 99%, of the cell population. Purity is measured by any appropriate standard method, for example, by fluorescence-activated cell sorting (FACS) using cell type-specific markers described herein. A population of cells used to cellularize an acellular valve structure or synthetic structure may be a mixture of two or more different cell types, each of which is isolated. For example, valves are colonized with a mixture of isolated myofibroblasts and isolated endothelial cells. An acellular or decellularized valve is one which is synthetic (not derived from a living organism) or one which has been treated to remove at least 85% of the cells with which it is naturally associated. Preferably, 90%, 95%, 99% or 100% of the cells with which the donor valve is associated in vivo are removed.
The myofibroblasts used to cellularize a valve matrix are obtained from a variety of tissue sources, e.g., cardiac, vascular, or dermal tissue. Preferably, the cells are derived from a human donor. Preferably, the cells are derived from histocompatible (e.g., autologous) mammalian heart leaflet interstitial tissue such as human heart leaflet interstitial tissue. Alternatively, the cells are derived from other tissue sources, e.g., dermal tissue, and cultured under conditions which promote a myofibroblast-like phenotype. The cells are syngeneic with respect to the intended recipient of the replacement valve.
To inhibit dedifferentiation of myofibroblasts, the cells are maintained in the presence of one or more cell signaling or growth factors which favor the leaflet myofibroblast phenotype (i.e., contractile and secretory function). The cells are maintained in static culture conditions or subjected to pulsatile flow culture conditions. Growth factors include basic fibroblast growth factor (bFGF). As is discussed above, the cells are cultured in endothelial cell-conditioned media or in physical contact with endothelial cells. Myofibroblasts may be cultured in the presence of a purified or recombinant growth factor. Preferably, the growth factor is derived from an endothelial cell, e.g., purified from endothelial cell conditioned media. The factor is purified using methods known in the art such as standard chromatographic techniques or recombinant cloning technology. A cell signaling factor is distinguished from a growth factor in that a signaling factor influences phenotype (e.g., secretory or contractile activity) rather than growth rate.
Conditioned media is fractionated by size and charge. The ability of each fraction to promote and maintain the myofibroblast phenotype is assessed using methods known in the art, e.g., qualititative evaluation by immunocytochemistry and histology to measure contractile and synthetic properties and quantitative evaluation using assays for matrix components including collagen, elastin and glycosaminoglycans. Proteins from the fraction(s) with the highest activity are purified and sequences using known methods. A secretory cell, e.g., one that has been genetically modified to produce a signaling factor, a growth factor or matrix component, is used in coculture with isolated myofibroblasts. For example, the secretory cell is of non-endothelial and non-myofibroblast origin.
Myofibroblast cells are cultured under pulsatile flow conditions to enhance production of type I collagen and minimize dedifferentiation. Cellularized valves cultured under such conditions assume the functional anatomy of a native valve. For example, the valve leaflets contain a monolayer of endothelial cells on the external layer and myofibroblasts in the inner layers. The leaflet interstitium contains a non-homogeneous matrix of one or more layers with myofibroblasts present in all layers and with collagen fibrils oriented in more than one direction. The cell culture conditions inhibit apoptosis of a myofibroblast that has been removed from a donor mammal, i.e., a harvested, cultured, transformed or transplanted myofibroblast. The culture method enhances viability and contractile activity of myofibroblasts in vitro.
Also within the invention is a genetically-modified myofibroblast. For example, the fibroblast is genetically modified to confer a myofibroblast phenotype, e.g., matrix synthetic capability, contractile capability The modified fibroblast produces increased levels of collagen I (compared to a normal, untreated fibroblast), fibronectin, or glycosaminoglycans. The cells may also be modified to express recombinant actin and myosin or heparin. Genetically-altered cells which have colonized a replacement heart valve are useful as an in vivo recombinant protein delivery system to deliver therapeutic polypeptides such as anticoagulant or antithrombotic agents.
A method of manufacturing an artificial heart valve includes the steps of (a) providing an acellular matrix, (b) seeding the matrix with isolated myofibroblasts; and (c) culturing the myofibroblasts under actual or biochemically simulated pulsatile flow conditions. Optionally, the matrix is seeded with additional cell types such as endothelial cells and/or secretory cells. The tissue culture media includes growth and cell signaling factors, e.g., those which are present in endothelial cell-conditioned media. Alternatively, factors are isolated from conditioned media, recombinant, or synthetic.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.
Other embodiments and features of the invention will be apparent from the following description thereof, and from the claims.